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WO1999039200A1 - Marqueurs moleculaires permettant le diagnostic de carcinome hepatocellulaire - Google Patents

Marqueurs moleculaires permettant le diagnostic de carcinome hepatocellulaire Download PDF

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WO1999039200A1
WO1999039200A1 PCT/US1999/001894 US9901894W WO9939200A1 WO 1999039200 A1 WO1999039200 A1 WO 1999039200A1 US 9901894 W US9901894 W US 9901894W WO 9939200 A1 WO9939200 A1 WO 9939200A1
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gene
cells
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expression
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Mark A. Feitelson
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Thomas Jefferson University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus

Definitions

  • HCC Primary hepatocellular carcinoma
  • HCC hepatitis B virus
  • HCV hepatitis C virus
  • HCC HCC is well established. Studies in Asia have shown that the incidence of this form of cancer over time is increased 100-fold in individuals with evidence of HBV infection as compared to non-infected controls (Podolsky, D.K. and K.J. Isselbacher. 1994. Harrison's Principles of Internal Medicine, pp. 1496-1497). More recent work in Europe and Japan has shown that
  • HCV is also linked to an increased risk of HCC.
  • any agent or factor that contributes to chronic, low-grade liver cell damage would make liver cell DNA more susceptible to damage and genetic alterations which can lead to carcinogenesis.
  • the mechanisms and steps responsible for the development of HCC have not been fully elucidated.
  • HBV hepatitis B x antigen
  • HBxAg binds to and functionally inactivates the tumor suppressor p53 (Feitelson, M.A. et al. 1993. Oncogene 8:1109-1117; Wang, X.W. et al. 1994. Proc. Natl. Acad. Sci. USA 91 :2230-2234; Truant, R. et al. 1995. J.
  • HBxAg/p53 staining and complex formation has also been shown to correlate with the development of liver tumors in a X transgenic mouse model with sustained high levels of HBxAg expression (Kim, CM. et al. 1991. Nature 351 :317-320; Koike, K. et al. 1994. Hepatology 19:810-
  • HBxAg is a trans-activating protein (Twu, J.S. and R.H. Schloemer. 1987. J. Virol. 61 :3448-3453; Rossner, M.T. 1992. J. Med. Virol. 36:101-117; Henkler, F. and R. Koshy. 1996. J. Viral Hepatitis 3:109-121). Even though virus DNA fragments - 3 -
  • HCC cells often contain the X region (Matsubara, K. and T.
  • HCC tumor cells form nontumor cells
  • HCC hepatitis or cirrhosis.
  • One screening tool has been alpha fetoprotein levels, where levels greater than 500 ⁇ g/L are found in 70-80% of patients with HCC (Podolsky, D.K. and K.J. Isselbacher. 1994. Harrison's Principles of Internal Medicine, pp. 1496-1497). The most common - 4 -
  • diagnostic tools are imaging with ultrasound, which can only detect the presence of visible tumors, and liver biopsy. Neither of these diagnostic tools is able to screen individuals for the risk of disease before tumors develop. In biopsy, it can be difficult to distinguish large cirrhotic nodules from well-differentiated HCC or low-grade dysplastic nodules from HCC.
  • the invention is a method for detecting hepatocellular carcinoma in liver tissue of a patient.
  • a liver tissue sample is obtained from the patient, and the level of expression of one or more marker genes in the sample is assessed.
  • the marker genes are differentially expressed in HBxAg[+] cells as compared with HBxAg[-] cells.
  • a reduction in the level of expression of one or more marker genes in the sample as compared to the expression level in noncancerous liver tissue is indicative of hepatocellular carcinoma in the sample.
  • the marker gene is selected from the group of genes expressing RNA transcripts which hybridize under conditions of high stringency to a nucleic acid probe selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.
  • the marker gene is selected from the group consisting of a gene which encodes the polypeptide of SEQ ID NO:27; a gene which encodes the polypeptide of SEQ ID NO:28; a gene which encodes the polypeptide of SEQ ID NO:29; a gene which encodes the polypeptide of SEQ ID NO:29; u-sui; human tubulin-folding cofactor E gene; human myeloblast KIAA0132 gene; and the human fetal heart gene, the cDNA of which is identified as GenBank accession number AA047006.
  • An example of high stringency hybridization conditions is hybridization at 4XSSC at 65°C, followed by washing in 0.1XSSC at 65°C for one hour.
  • Another example of high stringency hybridization conditions is hybridization in 50% formamide, 4XSSC at 55 °C.
  • the step of assessing the level of expression of the marker gene in the sample comprises contacting the sample with one or more probes which detect mRNA which is differentially expressed in HBxAg[+] cells as compared with
  • the step of assessing the level of expression of the marker gene by in the sample comprises assessing the level of expression of marker protein encoded by one or more marker genes. Detection of marker protein is accomplished by contacting the sample with one or more antibodies which bind marker proteins.
  • the invention provides a method for diagnosing hepatocellular carcinoma comprising the steps of obtaining a liver tissue sample from a patient, and assessing the level of expression of one or more marker genes in the tissue sample, which marker genes are differentially expressed in HBxAg[+] cells as compared - 6 -
  • HBxAgf-] cells The reduction of detectable expression of one or more marker genes in the sample is indicative of hepatocellular carcinoma.
  • the invention provides a method for identifying patterns in gene expression in a biological sample that are altered by hepatitis B x antigen comprising the steps of obtaining a biological sample; contacting said sample with a probe which detects an mRNA which is differentially expressed in HBxAg[+] cells as compared with HBxAg[-] cells; and detecting expression of a gene encoding said mRNA detected by the probe.
  • the steps for identifying alterations in gene expression patterns in the biological sample comprise contacting said sample with an antibody which detects a protein which is differentially expressed in HBxAg[+] cells as compared with HBxAg[-] cells; and detecting expression of a gene encoding the protein detected by the antibody.
  • HBV hepatitis B virus
  • HCC hepatocellular carcinoma
  • SSC standard saline citrate solution (0.15M saline containing 0.015M sodium citrate, pH 7)
  • “Expression” means, with respect to a gene, the realization of genetic information encoded in the gene to produce a functional RNA or protein. The term is thus used in its broadest sense, unless indicated to the contrary, to include either transcription or translation.
  • “Expression level”, with respect to a gene means a relative expression level as determined by comparison with the expression level of the gene in noncancerous tissue. An expression level may be "assessed” visually in a sample with the aid of a microscope, such as by considering the intensity of a stain for protein encoded by the gene of interest, or by considering the relative number of stained versus unstained cells in the sample.
  • Hybridization means the Watson-Crick base-pairing of essentially complementary nucleotide sequences (polymers of nucleic acids) to form a double-stranded molecule.
  • Marker gene means a gene which is differentially expressed in HCC tumor versus non-tumor tissue.
  • Marker protein means a protein which is encoded by a marker gene.
  • the present invention is a method for determining whether tissue from a biopsy represents HCC, based on detection of gene expression patterns in cells.
  • Studies of tumor and non-tumor pairs from patients demonstrate the differential expression of certain genes in tumor versus non-tumor tissue.
  • the genes are expressed in non-HCC tissue, but expression is substantially reduced or undetectable in HCC tumor tissue.
  • a reduction in expression of one or more of the marker genes in the tissue sample is diagnostic for the presence of HCC in the patient sample.
  • one or more of the marker genes may not be expressed or may be characterized by reduced expression level, such that the expression of the gene in the tissue sample as a whole is reduced.
  • the identification of such molecular markers provides a method for diagnosing HCC without relying on tissue morphology alone. This is the first time that molecular markers associated with chronic HBV infection have been shown to be useful in the diagnosis of HCC.
  • HepG2 is a differentiated cell line derived from a human hepatoblastoma.
  • the cell line HepG2X was generated by infection of HepG2 cells by replication defective recombinant retroviruses encoding the full length HBxAg polypeptide.
  • HepG2CAT cells were generated in the same manner by substituting the bacterial CAT gene for the HBV X gene in the transfection vector.
  • the HepG2X cells express the HBV X antigen (HBxAg[+]), while HepG2CAT cells do not (HBxAg[-]).
  • RNAs expressed in HepG2 cells are identified by PCR select cDNA subtraction. Briefly, the method consists of isolating whole cell RNA from HBxAg [+] and [-] HepG2 cells. Methods and kits for performing PCR select cDNA subtraction are well-known and commercially available, e.g., from Clontech, Palo Alto, CA. The RNA from HepG2X cells is subtracted from those in HepG2 cells, providing RNAs expressed in
  • RNAs were then reverse transcribed into DNA and then PCR amplified using random primers. In order to obtain RNAs expressed in HepG2, but not HepG2X cells, the opposite subtraction is carried out. These RT/PCR fragments were then cloned and either partially or fully sequenced. - 9 -
  • the clones represent fragments of genes whose expression is activated in HBxAg[+] cells compared to HBxAg[-] cells.
  • the clones represent genes whose expression is suppressed in HBxAg[+] cells compared to HBxAg[-] cells.
  • the fragment size given in Table 1 is considered approximate, as size was estimated visually from gels.
  • the cDNA fragments obtained from subtraction hybridization were used as probes for ISH of HepG2X and HepG2CAT cells, to verify that the probes obtained from PCR select cDNA subtraction actually represented differentially expressed genes in HepG2 compared to HepG2X cells. In all cases, the L probes hybridized to HepG2X cells. Little or no - 11 -
  • the cDNA fragments were either partially (L4, L7, L8, L15, L16 and C2) or completely (L11 , L12, L19 and C1) sequenced.
  • the sequences are as follows:
  • the full length cDNA containing the C2 sequence was obtained (from HepG2CAT cells) by 5' and 3' rapid amplification of cDNA ends (RACE) PCR using the MarathonTM cDNA Amplification Kit (Clontech, Palo Alto, CA). Briefly, one 3' and one 5' gene specific primers were synthesized. PCR was performed using these primers together with an adaptor primer to obtain the 3' or 5' cDNA specific products in separate amplification reactions. The products were cloned into pT7Blue T (Novagen, Inc., Madison, Wl) and sequenced. The - 12 -
  • HBxAg results in the altered expression of a protein whose function is associated with the regulation of translation. Further, HBxAg may contribute to hepato-carcinogenesis, in part, by altering gene expression at the level of translation initiation.
  • ISH was performed with the full set of ten individual probes (L4, L7, L8, L11 , L12, L15, L16, L19, C1 and C2) on tumor/nontumor paired samples from five HBV carriers with HCC, and on normal uninfected liver from two individuals.
  • the probes detected transcripts that were - 14 -
  • liver tissue is removed from an individual by conventional biopsy techniques which are well-known to those skilled in the art.
  • the test subject will be an HBV-infected individual.
  • the sample is generally collected by needle biopsy.
  • Procedures for liver needle biopsy are well-known in medicine. A mass may be apparent from either tactile examination of the patient, or upon imaging such as by ultrasound.
  • the needle biopsy should be taken at or near the site of the mass. Ultrasound guided percutaneous fine-needle biopsy procedures are known. See, e.g. , Polakow, Hepatogastroenterology 45:1829-30 (1998).
  • the biopsy sample may also be taken in connection with a surgical procedure in which the liver becomes accessible.
  • the expression level of the marker gene may serve as a convenient molecular marker to replace or augment conventional liver tissue examination, which largely relies on subjective criteria. This form of
  • molecular-based diagnosis can be performed more consistently than conventional pathological examination which is based upon subjective evaluations by expert pathologists.
  • Detecting the expression of the marker gene in the tissue sample comprises detecting RNA transcripts, particularly mRNA transcripts in the sample tissue, or detecting the corresponding marker gene product (protein) in the sample tissue.
  • the presence of the marker protein in the sample tissue is detected by an immunoassay whereby an antibody which binds the marker protein is contacted with the sampled tissue.
  • noncancerous liver tissue will be removed with the purported tumor tissue during biopsy.
  • the noncancerous (i.e., nontumor) cells will express the marker gene, and will provide a positive signal for the absence of HCC.
  • the noncancerous cells in the biopsied sample will serve as a convenient positive control.
  • control sample comprising liver cells from non-tumor liver tissue from the test subject, or non-tumor liver tissue from another (HBV-infected) individual.
  • the non-tumor sample should test positive for the expression of the marker gene.
  • Tissue samples may be considered as HCC-positive when the level of expression of one or more marker genes is reduced in the tissue sample as a whole, compared to the expression level in noncancerous liver tissue.
  • the overall reduction of marker gene expression in the liver sample may arise from a reduced but still detectable expression level in at least a portion of the cells of the sample, a complete loss of marker gene expression in some cells, or a combination of both.
  • the former may be apparent as a general lessening of stain intensity when the sample is treated with a stain for cells which express the marker gene. The latter may be observed as a complete absence of stain in the affected cells.
  • RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters by, e.g., the so-called "Northern" blotting technique.
  • the RNA is immobilized on the filters by - 16 -
  • RNA RNA probes complementary to the RNA in question See Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7, the disclosure of which is incorporated by reference.
  • the mRNA assay is carried out according to ISH.
  • ISH also known as "cytological hybridization", the in situ technique involves depositing whole cells or tissues onto a microscope cover slip and probing the nucleic acid content of the cell with a solution containing radioactive or otherwise labeled cDNA or cRNA probes.
  • ISH is described in more detail in U.S. Patent 5,427,916, the entire disclosure of which is incorporated herein by reference.
  • nucleic acid probes for the above RNA hybridization methods can be designed based upon the full length marker gene sequences described or referenced herein. Where the marker gene has yet to be identified with a known, full-length sequenced DNA, the corresponding cDNA fragment listed in Table 2 may be used as the probe.
  • the nucleic acid probe may be labeled with, e.g., a radionuclide such as 32 P, 14 C, or 35 S; a heavy metal; or a ligand capable of functioning as a specific binding pair member for a labeled ligand, such as a labeled antibody, a fluorescent molecule, a chemolescent molecule, an enzyme or the like.
  • a radionuclide such as 32 P, 14 C, or 35 S
  • a heavy metal or a ligand capable of functioning as a specific binding pair member for a labeled ligand, such as a labeled antibody, a fluorescent molecule, a chemolescent molecule, an enzyme or the like.
  • Probes may be labeled to high specific activity by either the nick translation method or Rig by et al., J. Mol. Biol. 113: 237-251 (1977) or by the random priming method, Fienberg et al., Anal. Biochem. 132: 6-13 (1983). The latter is the method of choice for synthesizing 32 P-labeled - 17 -
  • probes of high specific activity from single-stranded DNA or from RNA templates. Both methods are well-known to those skilled in the art and will not be repeated herein. By replacing preexisting nucleotides with highly radioactive nucleotides, it is possible to prepare 32 P-labeled DNA probes with a specific activity well in excess of 10 8 cpm/microgram according to the nick translation method. Autoradiographic detection of hybridization may then be performed by exposing filters on photographic film.
  • the random-primer method may be used to incorporate the " FTP analogue 5-(N-(N-biotinyl- epsilon-aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate into the probe molecule.
  • the thus biotinylated probe oligonucleotide can be detected by reaction with biotin binding proteins such as avidin, streptavidin, or anti-biotin antibodies coupled with fluorescent dyes or enzymes producing color reactions.
  • In situ hybridization is most conveniently carried out using a commercially available kit for labeling nucleic acid probes with, e.g. digoxigenenin/biotin as a label.
  • One such kit is available from Oncor, Gaithersburg, MD.
  • marker gene expression in cells of the patient tissue is determined by detecting the corresponding marker protein.
  • Western blots are run by spreading a protein sample on a gel, using an SDS gel, blotting the gel with a cellulose nitrate filter, and probing the filters with labeled antibodies.
  • immunohistochemical staining techniques a tissue sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the desired gene product.
  • the antibodies may be coupled to a visually detectable label, such as enzymatic labels, flourescent labels, luminescent labels, and the like.
  • tissue samples are obtained from patients and the samples are embedded and serially sectioned at 3-5 ⁇ per section.
  • the sections are fixed, mounted and dried according to conventional tissue mounting techniques.
  • the fixing agent may advantageously comprise formalin.
  • the embedding agent for mounting the specimen may comprise, e.g., paraffin.
  • the samples may be stored in this condition.
  • the samples are contacted with an immunoreagent comprising an antibody specific for a marker protein of interest.
  • the antibody may comprise a polyclonal or monoclonal antibody.
  • the antibody may comprise an intact antibody, or fragments thereof capable of specifically binding marker protein. Such fragments include, but are not limited to, Fab and F(ab') 2 fragments.
  • the term "antibody” includes both polyclonal and monoclonal antibodies. The term “antibody” means not only intact antibody molecules, but also includes fragments thereof which retain antigen binding ability.
  • Appropriate polyclonal antisera may be prepared by immunizing appropriate host animals with marker protein and collecting and purifying the antisera according to conventional techniques known to those skilled in the art.
  • Monoclonal antibody may be prepared by following the classical technique of Kohler and Milstein, Nature 254.493-497 (1975), as further elaborated in later works such as Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, R. H. Kennet et al., eds., Plenum Press, New York and London (1980).
  • Substantially pure marker protein for use as an immunogen for raising polyclonal or monoclonal antibodies may be conveniently prepared by recombinant DNA methods.
  • antibody against marker proteins can be raised by immunizing appropriate hosts with immunogenic fragments of the whole protein, particularly - 19 -
  • Hydrophilic regions which face the environment surrounding the protein, are most likely to contain antigenic sites. Such regions can be identified using standard computer programs.
  • the antibody either directly or indirectly bears a detectable label.
  • the detectable label may be directly attached to the primary anti-marker protein antibody. More conveniently, the detectable label is attached to a secondary antibody, e.g., goat anti-rabbit IgG, which binds the primary antibody.
  • the label may advantageously comprise, for example, a radionuclide in the case of a radioimmunoassay; a fluorescent moiety in the case of an immunofluorescent assay; a chemiluminescent moiety in the case of a chemiluminescent assay; or an enzyme which cleaves a chromogenic substrate, in the case of an enzyme-linked immunosorbent assay.
  • the detectable label comprises an avidin-biotin- peroxidase complex (ABC) which has surplus biotin-binding capacity. See Hsu et al., J. Histochem. Cytochem. 29:577-580, 1981.
  • the secondary antibody is biotinylated. Kits for staining proteins by the ABC method are commercially available (e.g., Vector Laboratories, Burlingame, CA). To determine the presence of marker protein antigen in the tissue section under analysis, the section is treated with primary antiserum against the antigen, washed, and then treated with the secondary antiserum.
  • the ABC method can be used for paraffin-embedded sections, frozen sections, and smears. Endogenous (tissue or cell) peroxidase may be quenched e.g. with H 2 0 2 in methanol.
  • the level of marker protein expression in samples may be compared on a relative basis to the expression in non-tumor liver tissue samples by comparing the stain intensities, or comparing the number of stained cells.
  • the diagnostic procedure described herein may take the form of detecting the expression of just one of the marker genes.
  • a mixture of probes (nucleic acid or antibody) targeting different marker genes may be utilized. This may be achieved by pooling two or more nucleic acid probes in the case of a nucleic acid hybridization assay, or by pooling two or more antisera in the case of a protein assay. By testing for the expression of multiple marker genes in this manner, it is expected that the sensitivity of the assay will be increased.
  • HepG2 cells a differentiated cell line derived from a human hepatoblastoma (Aden, D. P. et al. 1979. Nature 282:615-617; Knowles,
  • pSLXCMVneo The retroviral vector plasmid, pSLXCMVneo, was used to clone the HBV X gene (Valenzeula, P. et al. 1980. Animal Virus Genetics, Academic Press: New York, pp. 57-70) or the bacterial chloramphenicol acetyltransferase (CAT) gene sequences for these studies, as described (Duan, L.X. et al. 1995. Human Gene Ther.6:561-573). Briefly, pSLXCMV-
  • CAT was constructed by inserting a 726 bp Hindlll-BamHI fragment containing the CAT gene into the Hpal-Bglll site of the pSLXCMV polylinker.
  • PSLXCMV-FLAG-HBx was constructed by inserting a 920 bp Mlul-Bglll fragment of FLAG-HBx DNA into the Mlul-Bglll site of the pSLX- CMV polylinker. Recombinants were used to transform HB101. Minipreps were prepared and the DNA used for sequence analysis. - 22 -
  • PA317 cells/100 mm dish were transfected using standard calcium phosphate precipitation using 15 ⁇ g of pSLXCMV- FLAG-HBx or 15 ⁇ g of pSLXCMV-CAT.
  • the medium was removed and processed through a 0.45 ⁇ m filter to remove PA317 cells, and then used immediately for infection of HepG2 cells.
  • Five ml of recombinant retrovirus-enriched supernatant (5 x 10 6 CFU/ml, as assayed on NIH-3T3 cells) was used to infect 1 x 10 6 target HepG2 cells/100 mm dish in the presence of polybrene (8 ⁇ g/ml) for 24 hours.
  • Fresh virus supernatant was added after 24 and again after 48 hours so that the cells were exposed to virus for a total of 72 hours. All of these infections were carried out in log phase cultures. Cells were then passaged at 1 :2 and selected by incubation in G418 (800 ⁇ g/ml; GIBCO/BRL, Grand Island, NY) for 14 days in order to maximize the fraction of cells producing HBxAg or CAT. G418 colonies were then expanded in normal growth medium and used for analysis. The fourteen day selection in G418 had the effect of eliminating most of the uninfected cells.
  • the transfectants (HepG2-CAT and HepG2X) were evaluated as follows.
  • CAT assays were performed as described by Wang et al. (1994. Proc. Natl. Acad. Sci. USA 91 :2230-2234). Briefly, 1 x 10 7 HepG2-CAT cells in a 100 mm dish were lysed by addition of 0.9 ml of 1x report lysis buffer (Promega) for 15 minutes and harvested by scraping. Cells were pelleted and 180 ⁇ l of cell lysate was used for a standard CAT assay. After incubation with 4 C-chloramphenicol, acetylated forms were separated by thin-layer chromatography. Alternatively, lysates prepared from 5 x 10 6 - 23 -
  • HepG2X cells were assayed for the 17 kDa HBxAg by western blotting using a mixture well characterized rabbit anti-x peptide antibodies (Feitelson, M.A. and M.M. Clayton. 1990. Virology 177:367-371 ; Feitelson, M.A. etal. 1990. Gastroenterology 98: 1071 -1078).
  • Horseradish peroxidase conjugated goat anti-rabbit Ig (Accurate, Westbury, NY) and ECL substrate
  • CAT activity was present in HepG2CAT, but not in HepG2X cells.
  • HBxAg was present in lysates from HepG2X, but not from HepG2CAT cells.
  • PCR-select cDNA subtraction is reverse transcriptase (RT)/PCR based, and enriches for poly A + RNA (isolated using the Qiagen RNeasy total RNA kit; QIAGEN, Inc., Chatsworth, CA) from tissue culture cells ortissues.
  • the procedure involved ligating adaptors to some of the PCR products and conducting two rounds of subtractive hybridization against the PCR products from the cells in which the comparison were being made. The resulting products were then PCR amplified using primers which matched - 24 -
  • the HCC and surrounding nontumor liver tissues were obtained from two different sets of HCC patients.
  • the characteristic of the patients are set forth in Table 4:
  • HBsAg and HBcAg are, respectively, hepatitis B surface antigen and core antigen.
  • HBeAg is hepatitis B e antigen, a proteolytic fragment of HBcAg which is secreted as a free polypeptide into the blood of patients who replicate virus in the liver. HBeAg has thus been described as a surrogate marker for virus replication o Twenty-three paired tumor/nontumor samples came from as many
  • HBsAg positive Chinese carriers who had undergone surgery for the removal of their tumors. Most patients lived in and around Xi'an, China and were treated at the Fourth Military Medical University. Fourteen additional paired tumor/nontumor samples from as many patients were obtained from 5 South African patients. Half of these were HBV carriers (serum HBsAg positive) while the remaining patients, except for one, had evidence of past HBV infection (detectable anti-HBs and/or anti-HBc). Formalin fixed, paraffin embedded tissues, fresh frozen blocks, and -80°C snap frozen paired liver and tumor samples from individual patients were collected from o most patients, used for diagnostic purposes, and were then made available for these studies. Analogous pieces of uninfected human liver from two individuals were available to serve as controls.
  • Gene fragment C2 obtained from PCR select cDNA subtraction was used as a probe in in situ hybridization using the Oncor ISH and 5 digoxigenenin/biotin detection kits according to the instructions provided by - 26 -
  • Table 5 Summary of in situ hybridization for C2 probe in tumor /nontumor pairs for HCC patients from south Africa and China 3
  • ISH ln situ hybridization
  • the HCC and surrounding nontumor liver tissues used for analysis were obtained from five HBsAg positive Chinese carriers who had undergone surgery for the removal of their tumors. These patients were treated at the Fourth Military Medical University, Xi'an, China. Formalin fixed, paraffin embedded tissues, fresh frozen blocks, and -80°C snap - 28 -
  • T/NT tumor/nontumor
  • ISH ln situ hybridization
  • Synthetic peptides that represent probable antigenic determinants on each of the differentially expressed proteins were prepared by solid - 30 -
  • the peptides are identified in Table 7, below.
  • the The peptides were coupled by virtue of their free cysteine sulfhydryl (either in the peptide sequence or added to the carboxy or amino terminus where the native sequence did not contain a cysteine) to keyhole limpet hemocyanin (KLH; Sigma) using the coupling agent m-maleimidobenzyol- N-hydroxysuccinimide ester (MBS; Pierce) as described by Liu et al., Biochemistry 18:690-697 (1979).
  • Antisera generated from two or three peptide antigens of the same protein are pooled, and used in immunohistochemical staining assays as follows. Paired tumor/nontumor tissue samples from HBV- associated liver cancer HCC patients comprise the test samples.
  • Tissues are fixed in 10% formalin, embedded in paraffin and serially sectioned at 5m per section. Sections are then stained for individual differentially expressed proteins by the avidin-biotin complex (ABC) method (Hsu et al., J. Histochem Cytochem 29: 577-580) using a kit purchased from Vector Laboratories (Buriingame, CA). Staining is detected by addition of diaminobendizine (DAB) substrate, and the sections then counterstained with Mayer's hematoxylin. The degree of positive reaction is scored from 0 to +++. The grade 0 indicates no demonstrable antigen, + mild, ++ moderate and +++ dark staining.
  • ABS avidin-biotin complex

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Abstract

L'invention se rapporte à une méthode permettant de diagnostiquer un carcinome hépatocellulaire. Cette méthode vise à détecter des motifs différentiels d'expression génique qui sont engendrés par la présence de l'antigène X du virus de l'hépatite B dans les cellules hépatiques.
PCT/US1999/001894 1998-01-29 1999-01-27 Marqueurs moleculaires permettant le diagnostic de carcinome hepatocellulaire Ceased WO1999039200A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001013118A3 (fr) * 1999-08-18 2002-01-17 Oxford Glycosciences Uk Ltd Procedes et compositions permettant le diagnostic de l'hepatome
WO2001038579A3 (fr) * 1999-11-22 2002-02-14 Curagen Corp Methode d'identification d'agents toxiques utilisant l'expression genetique differentielle induite par les anti-inflammatoires non steroidiens (ains) dans le foie
WO2002029103A3 (fr) * 2000-10-02 2003-09-04 Gene Logic Inc Profils d'expression genetique dans le cancer du foie
WO2003027322A3 (fr) * 2001-09-25 2003-12-24 Japan President Univ Tokyo Genes et polypeptides associes aux carcinomes hepatocellulaires, et methode de detection de carcinomes hepatocellulaires
WO2004020661A3 (fr) * 2002-08-26 2005-03-24 Medlnnova Ges Fuer Medizinisch Systeme d'essai pour rechercher des principes actifs dans le cadre de dysfonctionnements des cellules nerveuses
WO2005106019A1 (fr) * 2004-04-29 2005-11-10 The University Of Hong Kong Surexpression de precurseur de granuline-epitheline (gep) en tant que cible destinee au diagnostic, au pronostic et au traitement de carcinome hepatocellulaire (chc)
CN100376687C (zh) * 2004-09-10 2008-03-26 香港中文大学 与肝细胞癌相关的乙型肝炎病毒基因组标志物
US7767392B2 (en) 2001-09-25 2010-08-03 Oncotherapy Science, Inc. Gene and protein relating to hepatocellular carcinoma and methods of use thereof
WO2018228029A1 (fr) * 2017-06-16 2018-12-20 上海易毕恩基因科技有限公司 Marqueur de gène pour l'utilisation dans la détection du cancer et utilisation de ce marqueur

Citations (1)

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US5534405A (en) * 1984-03-08 1996-07-09 The Scripps Research Institute Antibodies, diagnostic systems and methods for assaying HBxAg

Patent Citations (1)

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US5534405A (en) * 1984-03-08 1996-07-09 The Scripps Research Institute Antibodies, diagnostic systems and methods for assaying HBxAg

Non-Patent Citations (1)

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Title
FEITELSON M A, LING-XUN DUAN: "HEPATITIS B VIRUS X ANTIGEN IN THE PATHOGENESIS OF CHRONIC INFECTIONS AND THE DEVELOPMENT OF HEPATOCELLULAR CARCINOMA", AMERICAN JOURNAL OF PATHOLOGY., ELSEVIER INC., US, vol. 150, no. 04, 1 April 1997 (1997-04-01), US, pages 1141 - 1157, XP002920819, ISSN: 0002-9440 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001013118A3 (fr) * 1999-08-18 2002-01-17 Oxford Glycosciences Uk Ltd Procedes et compositions permettant le diagnostic de l'hepatome
US6852845B1 (en) 1999-11-22 2005-02-08 Curagen Corporation Method of identifying toxic agents using NSAID-induced differential gene expression in liver
WO2001038579A3 (fr) * 1999-11-22 2002-02-14 Curagen Corp Methode d'identification d'agents toxiques utilisant l'expression genetique differentielle induite par les anti-inflammatoires non steroidiens (ains) dans le foie
WO2002029103A3 (fr) * 2000-10-02 2003-09-04 Gene Logic Inc Profils d'expression genetique dans le cancer du foie
US7767392B2 (en) 2001-09-25 2010-08-03 Oncotherapy Science, Inc. Gene and protein relating to hepatocellular carcinoma and methods of use thereof
US7345156B2 (en) 2001-09-25 2008-03-18 Oncotherapy Science, Incorporated Hepatocellular carcinoma-related genes and polypeptides, and method for detecting hepatocellular carcinomas
WO2003027322A3 (fr) * 2001-09-25 2003-12-24 Japan President Univ Tokyo Genes et polypeptides associes aux carcinomes hepatocellulaires, et methode de detection de carcinomes hepatocellulaires
US7871763B2 (en) 2001-09-25 2011-01-18 Oncotherapy Science, Inc. Hepatocellular carcinoma-related genes and polypeptides, and method for detecting hepatocellular carcinomas
US8148080B2 (en) 2001-09-25 2012-04-03 Oncotherapy Science, Inc. Gene and protein relating to hepatocellular carcinoma and methods of use thereof
WO2004020661A3 (fr) * 2002-08-26 2005-03-24 Medlnnova Ges Fuer Medizinisch Systeme d'essai pour rechercher des principes actifs dans le cadre de dysfonctionnements des cellules nerveuses
WO2005106019A1 (fr) * 2004-04-29 2005-11-10 The University Of Hong Kong Surexpression de precurseur de granuline-epitheline (gep) en tant que cible destinee au diagnostic, au pronostic et au traitement de carcinome hepatocellulaire (chc)
CN1950521B (zh) * 2004-04-29 2012-04-25 香港大学 作为诊断、预后和治疗肝细胞癌(hcc)的目标的颗粒体蛋白-内皮素前体(gep)超表达
CN100376687C (zh) * 2004-09-10 2008-03-26 香港中文大学 与肝细胞癌相关的乙型肝炎病毒基因组标志物
WO2018228029A1 (fr) * 2017-06-16 2018-12-20 上海易毕恩基因科技有限公司 Marqueur de gène pour l'utilisation dans la détection du cancer et utilisation de ce marqueur

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